Pairing probiotics and prebiotics, methods for growth and use, separately and in combination

ABSTRACT

A method for growing probiotic organisms wherein the growth media includes prebiotics especially selected and prepared to be paired with the probiotic organisms being grown. The prebiotic formula is optimized to grow the desired probiotic organisms, as well as important byproducts of the growth process. Specialized freeze-drying buffers may also be paired with certain probiotic organisms for the freeze-drying process. Specific products may utilize selected probiotic organisms grown and supported with paired prebiotic formulas to promote and maintain physiological health in a subject.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/380,226, filed Apr. 10, 2019; which claims the benefit ofU.S. Provisional Patent Application Ser. No. 62/655,935, filed on Apr.11, 2018; and this application claims the benefit of U.S. ProvisionalPatent Application Ser. No. 62/854,490, filed on May 30, 2019; all ofwhich are hereby incorporated by reference in their entireties.

BACKGROUND The Field of the Invention

This invention relates to methods for producing and using probiotics,and more particularly to the pairing of particular probiotics andprebiotics to optimize growth and usefulness of the probiotics, both invivo and in vitro.

Background

Numerous products contain probiotics for a variety of purposes. Thesepurposes may include regulating digestion, improving and maintaininghealth in general, or improving specific physiological systems, as wellas other purposes. The probiotic Lactobacillus acidophilus is an exampleof the use of probiotics as a nutritional supplement, or as a componentin foods such as yogurts.

Generally, probiotics may be grown or cultured using a broth and/or anagar spread plate. There are various formulations for such a broth andvarious media used for such agar spread plates. These formulations arerelatively standardized for both broths and agar spread plates.

Methods for growing and culturing common probiotics, like Lactobacillusacidophilus, are generally established. For example, an original strainof the desired probiotic may be obtained and used an inoculationmaterial. The inoculation material may be grown in a fermenter (i.e., aliquid broth) and/or on a surface (i.e., an agar plate). This growthgenerally happens under pre-defined and monitored conditions. The grownprobiotic may then be harvested. After being harvested, the probioticmay be blended with other probiotics. The single probiotic or blend ofprobiotics may be preserved in some manner for transportation and/orstorage.

While there are various, useful probiotics that are found in nature, notall probiotics are available for commercial use. What is needed is animproved method or methods for culturing and harvesting more types ofprobiotics. Moreover, to get the full benefit of certain probiotics,those probiotics should be cultured or grown with the most appropriateprebiotics, or prebiotics specifically matched to optimize the growth ofthe desired probiotics. What is needed is an improved method or methodsfor producing or culturing various, useful probiotics to fully realizethe potential and benefits of those probiotics.

BRIEF SUMMARY OF THE INVENTION

In accordance with the foregoing, certain embodiments of a probioticproduct and/or method for production in accordance with the inventionmay provide probiotics that can be used for a variety of purposes,including without limitation, nutritional supplements, topicalapplications, and related uses and products.

There are a number of probiotic organisms that may be considered usefuland helpful to promote or maintain health in humans and animals. Suchprobiotics may include Bacteroides thetaiotaomicron, Bacteroidesfragilis, Bacteroides ovatus, Bacteroides uniformis, Faecalibacteriumprausnitzii, Akkermansia muciniphila, Eubacterium rectale, Collinsellaaerofaciens, Desulfovibrio piger, Clostridium symbiosum, Mycobacteriumvaccae, Eubacterium limosum, Butyricicoccus pullicaecorum, Roseomonasmucosa, Lactobacillus farciminis, Staphylococcus epidermis,Staphylococcus xylosus, Pediococcus acidilactici, Roseburia hominis, andAnaerostipes caccae. These probiotics, and methods for growing them, areprimarily described herein, but the methods and formulations describedherein may be applicable to other probiotic organisms.

A method for growing probiotic organisms, or growing unique probioticfermentations, may comprise a number steps. The method may includeselecting a probiotic organism to grow. The method may include selectinga growth broth for growing the probiotic organism, wherein the growthbroth includes a prebiotic formulation that is optimized for theselected probiotic organism. The method may include inoculating thegrowth broth with the probiotic organism. The method may include growingthe probiotic organism in the growth broth and harvesting the probioticorganism from the growth broth. The method may include selecting an agarspread plate for growing the probiotic organism, wherein the agar spreadplate media includes a prebiotic formula that is optimized for growingthe probiotic organism. The method may include inoculating the agarspread plate with the probiotic organism harvested from the growthbroth. The method may include growing the probiotic organism on the agarspread plate and harvesting the probiotic organism from the agar spreadplate.

A method may further include freeze-drying the harvested probioticorganism with a lyophilization reagent. The lyophilization reagent maybe comprised of one or more of the following: glucosamine, glutamine,sucrose, mannitol, trehalose, glycerol, inositol, raffinose, inulin,powdered skim milk, activated charcoal, soluble starch, collagen powder,chondroitin sulfate, glucosamine sulfate, guar, acacia, silica, andfructooligosaccharide P95.

A method as described herein may be used for growing any of theprobiotic organisms included, as well as other probiotic organisms. Amethod may include the use of a specific formulation of prebiotics thatare optimized for, or preferentially paired with, specific probioticorganisms. This pairing of certain prebiotics with certain probioticshelps promote better and faster growth of the desired probiotic. The useof certain, important prebiotics can promote the growth of a probioticorganism and specific byproducts of importance. Thus, a prebiotic, orprebiotic formulation, may be selected to be paired with a probioticorganism both for growing the probiotic organism and a desiredbyproduct, or group of byproducts, that may be helpful or important fora given purpose.

A method as described herein may be used for growing probiotic organismsthat are utilized in products designed to promote the health andmaintenance of a specific physiological system or function. For exampleand not by way of limitation, a product may include specific probioticorganisms that promote the health and maintenance of the immune system.Such a product may include a specific prebiotic formulation to supportthe specific probiotic organisms in vivo. Similarly, a product mayinclude specific probiotic organisms that promote and maintainneurological functions. Such a product may include a specific prebioticformulation to support the specific probiotic organisms in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of the present invention will become more fullyapparent from the following description and appended claims, taken inconjunction with the accompanying drawings and experimental data.Understanding that these drawings and date depict only typicalembodiments of the invention and are, therefore, not to be consideredlimiting of its scope, the invention will be described with additionalspecificity and detail through use of the accompanying drawings and datain which:

FIG. 1 illustrates a table providing results of measuring the amount ofcertain short chain fatty acids produced from certain probioticorganisms, where the values for short chain fatty acids are inmilligrams per milliliter measured from culture supernatant; and

FIG. 2 is a schematic diagram of an embodiment of a method for growingprobiotic organisms.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It will be readily understood that the components of the presentinvention, as generally described herein, could be arranged and designedin a wide variety of different configurations or formulations. Thus, thefollowing more detailed description of the embodiments of the system,product and method of the present invention, is not intended to limitthe scope of the invention, as claimed, but is merely representative ofvarious embodiments of the invention.

In one embodiment, a method for growing or culturing numerous probioticorganisms may include the following steps: selecting a probioticorganism, or a group of probiotic organisms, to grow; selecting a growthbroth, which may include a prebiotic formulation specially designed tohelp grow the probiotic organism selected; growing the probioticorganism in the growth broth; harvesting the probiotic organism from thegrowth broth; selecting a growth plate, which may include a prebioticformulation specially designed to help grow the probiotic organismselected; growing the probiotic organism on the growth plate; andharvesting the probiotic organism from the growth plate.

A probiotic organism, or a group of organisms, grown in this manner canbe freeze-dried after harvesting to prepare the organism for transferand/or storage.

The various steps in the process for growing a probiotic organism mayinclude variations at every level. For example, and not by way oflimitation, a growth broth may or may not include a prebiotic formulaspecially selected for the probiotic organism being grown. A prebioticformula may be specifically designed for one or more probioticorganisms, thereby allowing for numerous and varied formulations forprebiotic formulas. Also, the conditions for growing certain probioticorganisms can vary.

Generally, a probiotic organism grown in accordance with the disclosedmethod will be one or more of the following: Bacteroidesthetaiotaomicron, Bacteroides fragilis, Bacteroides ovatus, Bacteroidesuniformis, Faecalibacterium prausnitzii, Akkermansia muciniphila,Eubacterium rectale, Collinsella aerofaciens, Desulfovibrio piger,Clostridium symbiosum, Mycobacterium vaccae, Eubacterium limosum,Butyricicoccus pullicaecorum, Roseomonas mucosa, Lactobacillusfarciminis, Staphylococcus epidermis, Staphylococcus xylosus,Pediococcus acidilactici, Roseburia hominis, and Anaerostipes caccae.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 26 grams of tryptic soy broth, approximately4 grams of inulin, and approximately 0.4 grams of cysteine inapproximately 900 milliliters of water. Such a growth broth may be keptand/or used in a sealed one liter bottle. Such a growth broth may beused to grow a probiotic organism, for example and not by way oflimitation, Bacteroides fragilis, Bacteroides ovatus, Bacteroidesuniformis, Eubacterium limosum, Clostridium symbiosum, Parabacteroidesdistasonis, and Parabacteroides goldsteinii.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 26 grams of tryptic soy broth, approximately4 grams of inulin, approximately 0.4 grams of cysteine, andapproximately 3.6 milliliters of 50% glycerol in approximately 900milliliters of water. Such a growth broth may be kept and/or used in asealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation,Bacteroides thetaiotaomicron.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 26 grams of tryptic soy broth, approximately4 grams of inulin, approximately 2.5 grams of Middlebrook 7H9 broth, andapproximately 7.2 milliliters of 50% glycerol in approximately 900milliliters of water. Such a growth broth may be kept and/or used in asealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation,Mycobacterium vaccae. This probiotic organism may be grown aerobically.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 50 grams of MRS broth, approximately 0.1grams of glutathione, approximately 0.4 grams of cysteine, andapproximately 0.2 grams of uric acid in approximately 900 milliliters ofwater. Such a growth broth may be kept and/or used in a sealed one literbottle. Such a growth broth may be used to grow a probiotic organism,for example and not by way of limitation, Collinsella aerofaciens.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 50 grams of MRS broth in approximately 900milliliters of water. Such a growth broth may be kept and/or used in asealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation,Pediococcus acidilactici, Lactobacillus farciminis, and Lactobacillusvaginalis. These probiotic organisms may be grown aerobically oranaerobically.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 33 grams of brain heart infusion,approximately 0.1 grams of glutathione, approximately 0.4 grams ofcysteine, approximately 8 grams of inulin, approximately 4.5 grams ofyeast extract, and approximately 1.8 grams of sodium acetate inapproximately 900 milliliters of water. Such a growth broth may be keptand/or used in a sealed one liter bottle. Such a growth broth may beused to grow a probiotic organism, for example and not by way oflimitation, Faecalibacterium prausnitzii.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 7 grams of nutrient broth, approximately 4.5grams of tryptone, approximately 9 grams of yeast extract, approximately8 grams of inulin, approximately 4.5 grams of dextrose, approximately1.5 grams of dipotassium phosphate (K₂HPO₄), approximately 4.5milliliters of 20% tween 80, approximately 0.4 grams of cysteine, andapproximately 27 milliliters of a salt solution in approximately 900milliliters of water. The salt solution may be comprised ofapproximately 0.2 grams of calcium chloride (CaCl₂), approximately 0.24grams of magnesium sulfate (MgSO₄), approximately 1.0 grams ofmonopotassium phosphate (KH₂PO₄), approximately 10 grams of sodiumbicarbonate (NaHCO₃), and approximately 2.0 grams of sodium chloride(NaCl) in approximately one liter of water. Such a growth broth may bekept and/or used in a sealed one liter bottle. Such a growth broth maybe used to grow a probiotic organism, for example and not by way oflimitation, Eubacterium rectale, Anaerostipes caccae, and Butyricicoccuspullicaecorum.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 7 grams of nutrient broth, approximately 4.5grams of tryptone, approximately 9 grams of yeast extract, approximately8 grams of inulin, approximately 4.5 grams of dextrose, approximately1.5 grams of dipotassium phosphate (K₂HPO₄), approximately 4.5milliliters of 20% tween 80, approximately 0.4 grams of cysteine,approximately 0.1 grams of glutathione, and approximately 27 millilitersof a salt solution in approximately 900 milliliters of water. The saltsolution may be comprised of approximately 0.2 grams of calcium chloride(CaCl₂), approximately 0.24 grams of magnesium sulfate (MgSO₄),approximately 1.0 grams of monopotassium phosphate (KH₂PO₄),approximately 10 grams of sodium bicarbonate (NaHCO₃), and approximately2.0 grams of sodium chloride (NaCl) in approximately one liter of water.Such a growth broth may be kept and/or used in a sealed one literbottle. Such a growth broth may be used to grow a probiotic organism,for example and not by way of limitation, Eubacterium rectale andAnaerostipes caccae.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 7 grams of nutrient broth, approximately 4.5grams of tryptone, approximately 9 grams of yeast extract, approximately8 grams of inulin, approximately 4.5 grams of dextrose, approximately1.5 grams of dipotassium phosphate (K₂HPO₄), approximately 4.5milliliters of 20% tween 80, approximately 0.4 grams of cysteine,approximately 0.1 grams of glutathione, approximately 1.8 grams ofsodium acetate, approximately 0.9 grams of sodium lactate, andapproximately 27 milliliters of a salt solution in approximately 900milliliters of water. The salt solution may be comprised ofapproximately 0.2 grams of calcium chloride (CaCl₂), approximately 0.24grams of magnesium sulfate (MgSO₄), approximately 1.0 grams ofmonopotassium phosphate (KH₂PO₄), approximately 10 grams of sodiumbicarbonate (NaHCO₃), and approximately 2.0 grams of sodium chloride(NaCl) in approximately one liter of water. Such a growth broth may bekept and/or used in a sealed one liter bottle. Such a growth broth maybe used to grow a probiotic organism, for example and not by way oflimitation, Anaerostipes caccae, Eubacterium hallii, Intestinibacterbartlettii, and Subdoligranulum variabile.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 7 grams of nutrient broth, approximately 4.5grams of tryptone, approximately 9 grams of yeast extract, approximately8 grams of inulin, approximately 4.5 grams of dextrose, approximately1.5 grams of dipotassium phosphate (K₂HPO₄), approximately 4.5milliliters of 20% tween 80, approximately 0.4 grams of cysteine,approximately 0.1 grams of glutathione, approximately 0.2 grams ofammonium sulfate, and approximately 27 milliliters of a salt solution inapproximately 900 milliliters of water. The salt solution may becomprised of approximately 0.2 grams of calcium chloride (CaCl₂),approximately 0.24 grams of magnesium sulfate (MgSO₄), approximately 1.0grams of monopotassium phosphate (KH₂PO₄), approximately 10 grams ofsodium bicarbonate (NaHCO₃), and approximately 2.0 grams of sodiumchloride (NaCl) in approximately one liter of water. Such a growth brothmay be kept and/or used in a sealed one liter bottle. Such a growthbroth may be used to grow a probiotic organism, for example and not byway of limitation, Dorea longicatena.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 33 grams of brain heart infusion,approximately 1.0 grams of mucin, and approximately 0.4 grams ofcysteine in approximately 900 milliliters of water. Such a growth brothmay be kept and/or used in a sealed one liter bottle. Such a growthbroth may be used to grow a probiotic organism, for example and not byway of limitation, Akkermansia muciniphila.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 27 grams of tryptic soy broth, approximately4 grams of yeast extract, approximately 0.8 grams of sodium lactate,approximately 0.4 grams of magnesium sulfate, approximately 0.3 grams offerric ammonium sulfate-6H₂O, approximately 0.4 grams of cysteine, andapproximately 0.1 grams of glutathione in approximately 900 millilitersof water. Such a growth broth may be kept and/or used in a sealed oneliter bottle. Such a growth broth may be used to grow a probioticorganism, for example and not by way of limitation, Desulfovibrio piger.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 50 grams of MRS broth, approximately 0.4grams of cysteine, approximately 0.1 grams of glutathione, andapproximately 27 milliliters of a salt solution in approximately 900milliliters of water. The salt solution may be comprised ofapproximately 0.2 grams of calcium chloride (CaCl₂), approximately 0.24grams of magnesium sulfate (MgSO₄), approximately 1.0 grams ofmonopotassium phosphate (KH₂PO₄), approximately 10 grams of sodiumbicarbonate (NaHCO₃), and approximately 2.0 grams of sodium chloride(NaCl) in approximately one liter of water. Such a growth broth may bekept and/or used in a sealed one liter bottle. Such a growth broth maybe used to grow a probiotic organism, for example and not by way oflimitation, Roseburia hominis.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 7 grams of nutrient broth in approximately900 milliliters of water. Such a growth broth may be kept and/or used ina sealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation, Roseomonasmucosa. This probiotic may be grown aerobically.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 7 grams of nutrient broth and approximately4.5 grams of dextrose in approximately 900 milliliters of water. Such agrowth broth may be kept and/or used in a sealed one liter bottle. Sucha growth broth may be used to grow a probiotic organism, for example andnot by way of limitation, Roseomonas mucosa. This probiotic may be grownaerobically.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 27 grams of tryptic soy broth inapproximately 900 milliliters of water. Such a growth broth may be keptand/or used in a sealed one liter bottle. Such a growth broth may beused to grow a probiotic organism, for example and not by way oflimitation, Staphylococcus epidermidis. This probiotic organism may begrown aerobically or anaerobically.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 27 grams of tryptic soy broth andapproximately 7.2 milliliters of 50% glycerol in approximately 900milliliters of water. Such a growth broth may be kept and/or used in asealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation,Staphylococcus epidermidis. This probiotic organism may be grownaerobically or anaerobically.

In one embodiment, a growth broth, or liquid growth medium, may becomprised of approximately 27 grams of tryptic soy broth andapproximately 7.2 milliliters of 50% glycerol in approximately 900milliliters of water. Such a growth broth may be kept and/or used in asealed one liter bottle. Such a growth broth may be used to grow aprobiotic organism, for example and not by way of limitation,Staphylococcus xylosus. This probiotic organism may be grown aerobicallyor anaerobically.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 40 g/L tryptic soy agar, approximately 50ml/L defibrinated sheep blood, approximately 0.5 g/L cysteine, andapproximately 0.1 g/L glutathione. Such an agar spread plate may be usedto grow a probiotic organism, for example and not by way of limitation,Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides ovatus,Bacteroides uniformis, Eubacterium limosum, Clostridium symbiosum,Collinsella aerofaciens, Faecalibacterium prausnitzii, Eubacteriumrectale, Roseburia hominis, Anaerostipes caccae, Butyricicoccuspullicaecorum, Roseomonas mucosa, Staphylococcus epidermidis,Staphylococcus xylosus, Eubacterium hallii, Dorea longicatena,Intestinibacter bartlettii, and Subdoligranulum variabile.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 12 g/L agar and a growth broth(approximately 3 g/L) comprised of approximately 26 grams of tryptic soybroth, approximately 4 grams of inulin, approximately 2.5 grams ofMiddlebrook 7H9 broth, and approximately 7.2 milliliters of 50% glycerolin approximately 900 milliliters of water. Such an agar spread plate maybe used to grow a probiotic organism, for example and not by way oflimitation, Mycobacterium vaccae. This probiotic organism may be grownaerobically.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 70 g/L of MRS agar. Such an agar spreadplate may be used to grow a probiotic organism, for example and not byway of limitation, Pediococcus acidilactici, Lactobacillus farciminis,and Lactobacillus vaginalis. These probiotic organisms may be grownaerobically or anaerobically.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 12 g/L agar and a growth broth(approximately 3 g/L) comprised of approximately 33 grams of brain heartinfusion, approximately 1.0 grams of mucin, and approximately 0.4 gramsof cysteine in approximately 900 milliliters of water. A pour plate mayuse 8 g/L agar. Such an agar spread plate may be used to grow aprobiotic organism, for example and not by way of limitation,Akkermansia muciniphila.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 10 g/L agar and a growth broth(approximately 3 g/L) comprised of approximately 27 grams of tryptic soybroth, approximately 4 grams of yeast extract, approximately 0.8 gramsof sodium lactate, approximately 0.4 grams of magnesium sulfate,approximately 0.3 grams of ferric ammonium sulfate-6H₂O, approximately0.4 grams of cysteine, and approximately 0.1 grams of glutathione inapproximately 900 milliliters of water. A pour plate may use 10 g/Lagar. Such an agar spread plate may be used to grow a probioticorganism, for example and not by way of limitation, Desulfovibrio piger.

In one embodiment, an agar spread plate media, or agar spread plate, maybe comprised of approximately 52.5 g/L of reinforced clostridial agar inapproximately 900 milliliters of water. A pour plate may use 10 g/Lagar. Such an agar spread plate may be used to grow a probioticorganism, for example and not by way of limitation, Parabacteroidesdistasonis, and Parabacteroides goldsteinii.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe used to freeze-dry probiotic organisms that are grown and harvested.Freeze-drying reagents, or lyophilization reagents, may include thefollowing compounds alone or in combination: glucosamine, sucrose,mannitol, trehalose, glycerol, inositol, raffinose, inulin, powderedskim milk, activated charcoal, soluble starch, collagen powder,chondroitin sulfate, glucosamine sulfate, glutamine, guar, acacia,silica, and fructooligiosaccharide P95. A freeze-drying buffer, orlyophilization buffer, may be comprised of appropriate amounts of one ormore of the freeze-drying reagents in deionized water, which is thenadjusted to a pH of 7.0 with potassium hydroxide.

In one embodiment, a freeze-drying buffer may be made at least one dayprior to the freeze-drying process. A freeze-drying buffer may be storedovernight in an anaerobic chamber to allow dissolved oxygen to escapethe solution. The volume of a freeze-drying buffer used may be equal tothe volume of a cell pellet or probiotic organisms obtained aftercentrifugation.

In one embodiment, a freeze-drying buffer or media may be made withanti-oxidant and growth factors, for example and not by way oflimitation, cysteine, ascorbic acid, glutathione, uric acid, riboflavin,glutamic acid, sodium sulfite, quinones, alpha lipoic acid, resveratrol,vitamin A, and vitamin E.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe comprised of 10% mannitol, 12.5% sucrose, 2.5% trehalose, 1.0%glycerol, 10% skim milk, 0.02% cysteine, 0.01% ascorbic acid, 0.005%glutathione, 0.005% uric acid, and 2.0% charcoal in deionized water andadjusted to pH 7 with potassium hydroxide. The percentages provided maybe considered approximate percentages. This embodiment of afreeze-drying buffer will hereinafter be referred to as “Freeze-dryingBuffer A.” Freeze-drying Buffer A may be used with various probioticorganisms, for example and not by way of limitation, Mycobacteriumvaccae, Pediococcus acidilactici, Roseomonas mucosa, Lactobacillusfarciminis, Lactobacillus vaginalis, Staphylococcus epidermidis, andStaphylococcus xylosus.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe comprised of 10% mannitol, 12.5% sucrose, 2.5% trehalose, 1.0%glycerol, 10% skim milk, 0.2% cysteine, 0.1% ascorbic acid, 0.05%glutathione, 0.05% uric acid, and 2.0% charcoal in deionized water andadjusted to pH 7 with potassium hydroxide. The percentages provided maybe considered approximate percentages. This embodiment of afreeze-drying buffer will hereinafter be referred to as “Freeze-dryingBuffer B.” Freeze-drying Buffer B may be used with various probioticorganisms, for example and not by way of limitation, Bacteroidesthetaiotaomicron, Bacteroides fragilis, Bacteroides ovatus, Bacteroidesuniformis, Eubacterium limosum, Collinsella aerofaciens, Akkermansiamuciniphila, and Roseburia hominis.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe comprised of 10% mannitol, 10% inulin, 5% short chain FOS, 10%soluble starch, 2.5% trehalose, 1.0% glycerol, 2.0% cysteine, 0.5%ascorbic acid, 0.5% glutathione, 0.1% uric acid, and 6.0% charcoal indeionized water and adjusted to pH 7 with potassium hydroxide. Thepercentages provided may be considered approximate percentages. Thisembodiment of a freeze-drying buffer will hereinafter be referred to as“Freeze-drying Buffer C.” Freeze-drying Buffer C may be used withvarious probiotic organisms, for example and not by way of limitation,Clostridium symbiosum, Faecalibacterium praunsnitzii, Eubacteriumrectale, Anaerostipes caccae, Butyricicoccus pullicaecorum, Eubacteriumhallii, Dorea longicatena, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Intestinibacter bartettii, and Subdoligranulum variable.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe comprised of 10% mannitol, 12.5% sucrose, 2.5% trehalose, 10%collagen powder, 1.0% chondroitin sulfate, 1.0% glycerol, 2.0% cysteine,0.5% ascorbic acid, 0.5% glutathione, 0.1% uric acid, and 6.0% charcoalin deionized water and adjusted to pH 7 with potassium hydroxide. Thepercentages provided may be considered approximate percentages. Thisembodiment of a freeze-drying buffer will hereinafter be referred to as“Freeze-drying Buffer D.” Freeze-drying Buffer D may be used withvarious probiotic organisms, for example and not by way of limitation,Desulfovibrio piger.

In one embodiment, a freeze-drying buffer, or lyophilization buffer, maybe comprised of 10% mannitol, 10% inulin, 5% short chain FOS, 10%soluble starch, 2.5% trehalose, 1% glycerol, 5% cysteine, 1% ascorbicacid, 5% glutathione, 1% uric acid, 1% alpha lipoic acid, 1% resveratol,and 6.0% charcoal in deionized water and adjusted to pH 7 with potassiumhydroxide. The percentages provided may be considered approximatepercentages. This embodiment of a freeze-drying buffer will hereinafterbe referred to as “Freeze-drying Buffer E.” Freeze-drying Buffer E maybe used with various probiotic organisms, for example and not by way oflimitation, Faecalibacterium prausnitzii.

In one embodiment, a dilution buffer, or anti-oxidant resuspensionsolution, may be utilized for plating lyophilized bacteria and/orobtaining plate counts and estimating cfu/gram (colony-forming unit pergram of sample). In one embodiment, a dilution buffer, or anti-oxidantresuspension solution, may be comprised of 0.2% cysteine, 0.1% ascorbicacid, 0.05% glutathione, 0.05% uric acid, and 0.0425% monopotassiumphosphate (KH₂PO₄) monopotassium phosphate (KH₂PO₄) in deionized waterand adjusted to pH 7 with potassium hydroxide solution.

Culture conditions for various probiotic organisms have been determinedexperimentally.

In one embodiment, various probiotic organisms, for example and not byway of limitation, Bacteroides thetaiotaomicron, Bacteroides fragilis,Bacteroides ovatus, Bacteroides uniformis, Eubacterium limosum,Collinsella aerofaciens, Akkermansia muciniphila, Anaerostipes caccae,and Desulfovibrio piger require 48-96 hours of incubation at 37° C. toachieve the desired growth. The addition of anti-oxidants during mediapreparation may be required, but no special anaerobic conditions arerequired for growth in liquid media, or growth broth. The liquid mediabottles should be capped to prevent contamination and to limit airflowto the growing probiotic cultures. Disturbance of the liquid media, suchas swirling, should be avoided to limit oxygen rich air being mixed withthe liquid media.

Centrifugation and freeze-drying processes can be performed in anaerobic environment if done quickly, for example, by mixing thecentrifuged pellet with a freeze-dry buffer immediately after pouringoff the supernatant and then placing the suspension at −20° C. It maytake approximately one hour to fully freeze the suspension. Then thesuspension can be placed in the lyophilizer.

Spread plating or pour plating techniques can be used for theseprobiotic organisms, provided that the dilution buffer, or antioxidantresuspension solution, is used. Plates should be put in an anaerobicchamber or airtight box containing anaerobic gas producing packets.Growth of colonies on plates typically requires 24-72 hours at 37° C.

In one embodiment, various probiotic organisms, for example and not byway of limitation, Clostridium symbiosum, Faecalibacterium prausnitzii,Eubacterium rectale, Roseburia hominis, Anaerostipes caccae,Butyricicoccus pullicaecorum, Eubacterium hallii, Dorea longicatena,Parabacteroides distasonis, Parabacteroides goldsteinii, Intestinibacterbartettii, and Subdoligranulum variable require 24-48 hours ofincubation at 37° C. to achieve desired growth. These probioticorganisms grow poorly unless incubated in an oxygen-free environment.Oxygen rich air must be evacuated from liquid media bottles beforeplacing them in an anaerobic chamber.

For centrifugation, liquid cultures must be poured into airtightcentrifuge bottles and sealed in an anaerobic chamber. The centrifugebottles can then be removed from the chamber to undergo centrifugation.They must be placed back in the anaerobic chamber before opening to pouroff the supernatant. The cell pellets are then suspended in freeze-drybuffer that has been sitting in the anaerobic chamber overnight, or forapproximately twelve hours, to help pull off oxygen dissolved in thebuffer. The cell suspension is then sealed in an airtight container andplaced at −20° C. until lyophilized in aerobic conditions.

All plating techniques must be performed in the anaerobic chamber.Growth of colonies on plates typically requires 24-48 hours at 37° C.

In one embodiment, various probiotic organisms, for example and not byway of limitation, Mycobacterium vaccae, Pediococcus acidilactici,Roseomonas mucosa, Lactobacillus farciminis, Lactobacillus vaginalis,Staphylococcus epidermidis, and Staphylococcus xylosus require 24-48hours of incubation at 37° C. to achieve desired growth, except that M.vaccae may require 5-8 days. These probiotic organisms may be culturedaerobically. Aeration of liquid media, or growth broth, using a stirplate and a stir bar improves growth of M. vaccae, R. mucosa, S.epidermidis, and S. xylosus.

Centrifugation, any freeze-drying process, and plate growth techniquesmay all occur in an aerobic environment. Colonies on plates aretypically observed in 24-48 hours at 37° C., except that M. vaccae maytake 3-5 days.

Prebiotic formulations may be used to supplement, support, and optimizeprobiotic growth, which process may be referred to as synbiotics. Inorder to facilitate growth of a given probiotic organism, the correct ormatching prebiotic is crucial. Since probiotic organisms vary in genusand species, specific prebiotic preparations or formulations may berequired. Some prebiotics colonize the lumen and others the mucuslayers. Many prebiotics are butyrate-producing Firmicutes members.

Prebiotics often enhance mucosal butyrate, which has been shown topossibly stimulate the release of mucosal butyrate producers towards thelumen. A process connected with that may affect the intestinalinflammation and consumption of prebiotic compounds.

Some probiotic organisms require other special conditions likeco-culturing with another commensal organism. For example and not by wayof limitation, Bacteroides fragilis produces GABA (Gamma-AminobutyricAcid), a metabolite required by a second organism. Therefore, the twoorganisms may be considered co-dependent.

Various techniques may be utilized to introduce a prebiotic, or aprebiotic formulation, to a method for growing a certain probioticorganism, or multiple probiotic organisms. In one embodiment, a growthmedia may be exogenously supplemented with the needed metabolite. Inanother embodiment, the supernatant of one organism may be used tosupplement the growth media used for growing another organism ofinterest. For example and not by way of limitation, growth ofFaecalibacterium prausnitzii may require special supplementation of oneor more specific vitamin K metabolites.

Table 1 below describes a prebiotic formulation that may be used withprobiotic organisms including Faecalibacterium prausnitzii,Mycobacterium vaccae, and Lactobacillus farciminis.

TABLE 1 Prebiotic mg/ser Galacto-oligosaccharide 3000.0Isomalt-oligosaccharide 3000.0 IgY-immunoglobulin 100.0 Deoxynojirimycinpolysaccharide (DPM) 500.0 Lactoferrin 50.0 Guggulipids 500.0 Sorghum2000.0 Pectin 1000.0 Fructooligosaccharide (FOS) 1000.0 Blueberry powder1000.0 Black currant 1000.0 Lions mane mushroom 500.0 Cordycepsmilitarus 500.0 Karaya gum 500.0 Pomegranate (ellagitanin) 500.0Geranium thunbergii (dehydroellagitanin) 500.0 Phyllanthus muellerianus(Geraniin and furosin) 500.0 Raspberry extract (gallic ellagic) 500.0Total 16650.0

The amounts in Table 1 may be considered approximations. The prebioticformulation represented in Table 1 may be hereinafter referred to as“Prebiotic Formula Alpha.”

Table 2 below describes a prebiotic formulation that may be used withprobiotic organisms including Eubacterium limosum, Bacteroidesthetaiotaomicron, Lactobacillus vaginalis, and Bacteroides uniformis.

TABLE 2 Prebiotic mg/ser Glycomacropeptide 1000.0 Xylo-oligosaccharide1000.0 Glucomannan 1000.0 Inulin; sprouted greens 1000.0 Galactomannan1000.0 Blueberry powder 1000.0 Thymus 1000.0 Mulberry leaves 1000.0Myo-inositol 1000.0 Modified citrus pectin 1000.0 Tapioca 500.0Glucosaminoglycan 1000.0 Guar sun-fiber 500.0 Oat oligosaccharide 500.0Clitoria ternatea (blue pea flower) 500.0 Cranberry extract 500.0Bearberry (Arctostaphylos uva-ursi) 500.0 Total 14000.0

The amounts in Table 2 may be considered approximations. The prebioticformulation represented in Table 2 may be hereinafter referred to as“Prebiotic Formula Beta.”

Table 3 below describes a prebiotic formulation that may be used withprobiotic organisms including Bacteroides ovatus, Anaerostipes caccae,Staphylococcus xylosus, Staphylococcus epidermidis, and Roseomonasmucosa.

TABLE 3 Prebiotic mg/ser 2′fructosyllactose 2000.0Isomato-oligosaccharide 1000.0 Fructooligosaccharide Yaconroot/Beneo/innulin 1000.0 Galactooligosaccharide 1000.0 Rutin 1000.0 Oatoligosaccharide 1000.0 Sialylated bovine milk oligosaccharides (S-BMO)1000.0 IgY immunoglobulin 1000.0 Beta glucan 1000.0 Maca 1000.0Lactooligosaccharide 1000.0 Medium Chain Triglycerides 500.0Phosphatidyl choline 500.0 Rose hips/organic green banana 500.0 Citrusbioflavonoids 500.0 Colostrum 500.0 Total 14500.0

The amounts in Table 3 may be considered approximations. The prebioticformulation represented in Table 3 may be hereinafter referred to as“Prebiotic Formula Gamma.”

Table 4 below describes a prebiotic formula that may be used withprobiotic organisms including Collinsella aerofaciens, Bacteroidesovatus, Bacteroides uniformis, Clostridium symbiosum, Bacteroidesfragilis, and Roseburia hominis.

TABLE 4 Prebiotic mg/ser Galacto-oligosaccharide 2000.0Mannan-oligosaccharide 1000.0 Resveratrol 1000.0 Hesperetin 1000.0Arabinoxylan oligosaccharides 1000.0 Dragon fruit 1000.0 Beechwoodextract xylan 1000.0 Arabinogalactan (Larix laricina) 1000.0 DAOantihistamine 1000.0 Xanthan gum 1000.0 Resistant starch 1000.0 Baobabfruit 500.0 Kongorobi berry 1000.0 Resveratrol/Pterostilbene 500.0Curcumin 500.0 Indian Tinospora (Tinospora cordifolia)(stem and root)500.0 Poria mushroom 500.0 Total 15500.0

The amounts in Table 4 may be considered approximations. The prebioticformulation represented in Table 4 may be hereinafter referred to as“Prebiotic Formula Delta.”

The various prebiotic formulations may be utilized with a liquid mediaor growth broth. The prebiotic formulation selected and paired with agiven probiotic organism may constitute approximately 1%-15% of thegrowth broth. Similarly, the various prebiotic formulations may beutilized with an agar spread plate. The prebiotic formulation selectedand paired with a given probiotic organism may constitute approximately1%-15% of the agar spread plate media.

Prebiotic ingredients used in prebiotic formulations can many differenttypes of compounds. For example and not by way of limitation, complexplant oligosaccharides may include the following:Galacto-oligosaccharide; Isomalt-oligosaccharide;Mannan-oligosaccharide; Apple pectin oligosaccharide;Xylo-oligosaccharide; and Fructo-oligosaccharide-inulin. These complexplant oligosaccharides have been shown increase caecal mucin levels byapproximately six-fold and correspond with even higher butyrate levelsand higher abundances of the same mucosal butyrate producers.

Other complex plant oligosaccharides include Oat-oligosaccharides andArabinoxylan-oligosaccharides (AXOS), which have been shown to be apromising class of prebiotics that stimulate the growth ofBifidobacterium longum, an acetate producer to stimulate Eubacteriumrectale, an acetate-converting butyrate producer.

Another complex plant oligosaccharide is Deoxynojirimycin polysaccharide(DPM), which has been shown to increase the Bacteroides to Firmicutesratios, significantly inhibiting the growth of Prevotella, andincreasing the relative abundance of Bacteroides, Lactobacillus,Bifidobacterium, and Akkermansia in mice.

Animal based oligosaccharides may include the following: N-acetylglucosamine-amino glycan Lactulose-saccharide; IgY-immunoglobulin; andSialylated bovine milk oligosaccharides (S-BMO).

Other complex prebiotic compounds may include the following:Cyclodextrin-saccharide; Mulberry-cyanadins; Maca-oligosaccharides;polyphenols; Rutin-flavonoid (E. limosum specifically metabolizedflavonoids); mushroom-polysaccharides; aminoglycans; Shilajit (organiccompounds); Resveratrol; and Pterostilbene.

The criteria for pairing or partnering certain probiotic organisms withspecific prebiotics is driven by the metabolites that are produced bythe probiotic organisms. For example and not by way of limitation, themetabolites produced by probiotic organisms that may be evaluated topair or partner those probiotic organisms with specific prebiotics mayinclude the following: small chain fatty acids, such as butyric acid,acetic acid, propionic acid, hexanoic acid, and the like; organic acids,such as lactic acid, malic acid, citric acid, and the like;bacteriocins, which are natural antimicrobials produced by probioticorganisms. The research and analysis required to properly partnerprobiotic organisms and prebiotic compounds can take years to complete.

FIG. 1 provides an example of the research conducted and evaluated toproperly partner probiotic organisms and prebiotic compounds based onthe production of short chain fatty acids produced in selected strainsof probiotic organisms. The values for short chain fatty acids in FIG. 1are in milligrams per milliliter measured from culture supernatant.

In one embodiment, a phage may be used as a terrain biotic. For exampleand not by way of limitation, terrain biotics may include Streptococcusmutans, Actinomyces naeslundi, Cutibacterium acnes, Porphyromonasgingivalis, Treponema denticola, Fusobactium nucleatum, Aggregatibacteractinomycetemcomitans, and Tannerella forsythia.

Referring to FIG. 2, one embodiment of a method 10 for producingprobiotic organisms may include the steps of selecting a probioticorganism to grow 15, selecting a prebiotic formula 20 to pair with theprobiotic organism selected, selecting a growth broth 25, inoculatingthe growth broth 30, growing the probiotic organism 35, harvesting theprobiotic organism from the growth broth 40, selecting a growth plate45, inoculating the growth plate 50, growing the probiotic organism onthe growth plate 55, harvesting the probiotic organism from the growthplate 60, and freeze-drying the probiotic organism 65.

The step of selecting a probiotic organism to grow 15 can be based onnumerous factors. The uses, properties, and metabolites of a givenprobiotic organism may be considered. Generally, the process ofbeginning to grow a probiotic organism will start with a pure strain ofthe organism, but it is possible to grow multiple probiotic organismstogether. However, more care should be taken to select organisms thatcan be grown using the same growth media, as well as the same prebioticformulations, to optimize growth of all probiotic organisms selected.

The step of selecting a prebiotic formula, or formulation, 20 to growthe selected probiotic organism should be based primarily on theprobiotic organism selected. The intention is to match or partner theprebiotics with the probiotic organism as best as can be done to promoteoptimal growth of the probiotic organism. For example and not by way oflimitation, if Mycobacterium vaccae is the probiotic organism selectedto be grown, Prebiotic Formula Alpha would be an optimal choice forprebiotics to pair with the probiotic organism during the growthprocess.

The step of selecting a growth broth 25, or selecting a liquid media forgrowth 25, may include a number of factors. A growth broth can beselected based on the particular probiotic organism to be grown, andthere are certain growth broths that can be used to grow specificprobiotic organisms. Also, a growth broth may include at least some of aprebiotic formulation to promote the growth process. A growth broth maybe augmented to include a prebiotic formula where the prebiotic formulais approximately 1%-15% of the growth broth media. For example and notby way of limitation, if Bacteroides fragilis is the probiotic organismto be grown, the growth broth used (and previously described) may beaugmented to be approximately 10% Prebiotic Formula Delta.

The step of inoculating the growth broth 30 is generally performed byintroducing the selected probiotic organism into the selected andprepared growth broth by any appropriate means.

The step of growing the probiotic organism in the growth broth 35 isaccomplished by any appropriate means. For example and not by way oflimitation, if Bacteroides fragilis is the probiotic organism to begrown, the growth broth may be kept in a sealed bottle and allowed toincubate for 48-96 hours at 37° C. For example and not by way oflimitation, if Mycobacterium vaccae is the probiotic organism to begrown, the growth should take place anaerobically.

The step of harvesting the probiotic organism from the growth broth 40is generally accomplished by any appropriate means that are consistentwith the requirements for the probiotic organism. For example and not byway of limitation, if Mycobacterium vaccae is the probiotic organism tobe grown, this harvesting process should take place anaerobically.

The step of selecting a growth plate 45, or selecting an agar spreadplate for growing the selected probiotic organism 45, may includeconsideration of multiple factors. The type of media used for the growthplate should be compatible with the probiotic organism to be grown.Also, the media of the growth plate may include a prebiotic formulationto promote growth of the probiotic organism. For example and not by wayof limitation, if Bacteroides thetaiotaomicron is the probiotic organismto be grown, the growth plate used, or agar spread plate used (andpreviously described), may be augmented to include approximately 5%Prebiotic Formula Beta.

The step of inoculating the growth plate 50, or inoculating and/orpreparing the agar spread plate 50, is generally performed byintroducing the probiotic organism harvested from the growth broth 40onto the surface of the selected and prepared growth plate by anyappropriate means. For example and not by way of limitation, ifRoseburia hominis is the probiotic organism to be grown, the inoculatingand/or preparing the growth plate should take place anaerobically.

The step of growing the probiotic organism on the growth plate 55, orgrowing the probiotic organism on the agar spread plate 55, isaccomplished by any appropriate means. For example and not by way oflimitation, if Bacteroides fragilis is the probiotic organism to begrown, the growth plate may be allowed to incubate for 24-72 hours at37° C. For example and not by way of limitation, if Mycobacterium vaccaeis the probiotic organism to be grown, the growth should take placeanaerobically.

The step of harvesting the probiotic organism from the growth plate 60is generally accomplished by any appropriate means that are consistentwith the requirements for the probiotic organism. For example and not byway of limitation, if Mycobacterium vaccae is the probiotic organism tobe grown, this harvesting process should take place anaerobically.

The step of freeze-drying the probiotic organism 65 may includeconsideration of a variety of factors. There may be times when thefreeze-drying step is not required or desired. The probiotic organismmay require preparation for storage and/or delivery. Also, a speciallyprepared buffer for the freeze-dry process, or freeze-drying buffer, maybe utilized for lyophilization of a specific probiotic organism. Forexample and not by way of limitation, if Clostridium symbiosum is theprobiotic organism that has been grown, then Freeze-drying Buffer C maybe used to more appropriately freeze-dry that probiotic organism.

Similar to how there are numerous, possible combinations of probioticswith suitable or optimal prebiotics, there are also numerous, possiblecombinations of probiotics that can be used to help with or benefitselected physiological functions. Moreover, each probiotic selected forsuch a combination can be accompanied by its most advantageousprebiotics. For example, one or more probiotics can be selected for andprovided in a product for supporting neurological functions, immunesystem functions, female functions, male functions, and metabolicfunctions, as well as others.

Certain microorganisms are capable of influencing the central nervoussystem. This process is sometimes described as the “microbiota-gut-brainaxis.” There are a number of mechanisms that have been identified asmediating this phenomenon, including the vagal nerve, the immune system,and neurotransmitters. There may be a number of links between thegastro-intestinal microbiome and the brain related to importantphysiological functions, including neurotransmitter production, entericnervous system hormones, central nervous system development, mood,anxiety, stress and others.

In one embodiment, a combination of cultured organisms designed tobenefit the health of the microbiota-gut-brain axis may include one ormore of the following probiotic organisms: Enterococcus faecium,Bifidobacterium adolescentis, Faecalibacterium prausnitzii,Mycobacterium vaccae, Lactobacillus plantarum, Bifidobacterium longum,Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillusfarciminis, Propionibacterium shermanii, Bifidobacterium infantis,Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillusdelbrueckii, Bacteroides fragilis, Bifidobacterium lactis, andLactobacillus rhamnosus A. These probiotic organisms may be provided inan approximate CFU range of 1.00E+06-1.00E+11.

There is a distinct advantage for these microbiota-gut-brain axisprobiotic organisms if they are provided with the proper prebiotics,especially if it is desired that the probiotics remain in thegastro-intestinal tract for an extended amount of time. A prebiotic thatmay be used to feed these microbiota-gut-brain axis probiotic organismsis shown in Table 5.

TABLE 5 Prebiotic mg/dose Galacto-oligosaccharide 500-5000Isomalt-oligosaccharide/Maltosyl-isomaltooligosaccharide 500-5000IgY-immunoglobulin 25-500 Deoxnoiirimycin polysaccharide (DPM) 500-5000Lactoferrin 25-500 Commiphora mukul (Guggulipids) 50-60  Sorghum bicolor500-5000 Pectin (Citrus or Rose Hips) 500-5000 FOS(fructo-oligosaccharide) 500-5000 Blueberry (Vaccinium corymbosum)powder 500-5000 Black currant (Ribes nigrum) 500-5000 Lion's manemushroom (Hericium erinaceus) 500-5000 Cordyceps militarus 500-5000Karaya gum (Sterculia urens) 500-5000

The amounts in Table 5 may be considered approximations. The prebioticformulation represented in Table 5 may be hereinafter referred to as“Prebiotic Formula Epsilon.”

In one embodiment, the prebiotics in Table 6 may be used in vivo tosupport the following probiotic organisms: Faecalibacterium prausnitzii,Mycobacterium vaccae, Lactobacillus farcimins, Parabacteroidesgoldsteinii, and Subdoligranulum variable.

TABLE 6 Prebiotic mg/dose Galacto-oligosaccharide 500-5000Isomalt-oligosaccharide/Maltosyl-isomaltooligosaccharide 500-5000IgY-immunoglobulin 25-500 Deoxnoiirimycin polysaccharide (DPM) 500-5000Lactoferrin 25-500 Commiphora mukul (Guggulipids) 50-60  Sorghum bicolor500-5000 Pectin (Citrus or Rose Hips) 500-5000 FOS(fructo-oligosaccharide) 500-5000 Blueberry (Vaccinium corymbosum)powder 500-5000 Black currant (Ribes nigrum) 500-5000 Lion's manemushroom (Hericium erinaceus) 500-5000 Cordyceps militarus 500-5000Karaya gum (Sterculia urens) 500-5000 Saffron (Crocus sativus) 25-500Ellagitannins-punicalagins-Pomegranate PPE 25-500 (Punica granatum)Geranium thunbergii (dehydroellagitannin) 25-500 Phyllanthusmuellerianus (Geraniin and furosin) 25-500 Rasberry (Rubus idaeus)(gallic ellagic) 25-500 Gold/Green kiwi (Actinidia chinensis andActinidia 500-5000 deliciosa)

The amounts in Table 6 may be considered approximations. The prebioticformulation represented in Table 6 may be hereinafter referred to as“Prebiotic Formula Zeta.”

One of the central functions of a person's microbiome is to train ourimmune system. The immune system is co-dependent on the microbiome forits education and its ongoing maintenance. A number of probioticorganisms have been identified as directly modulating our immunefunction. In one embodiment, a unique combination of probiotics may bedesigned around both our innate and adaptive immune systems. Properimmune modulating is dependent on prebiotics to successfully colonizethe gastrointestinal tract.

In one embodiment, a combination of cultured organisms designed tobenefit the health of the immune system may include one or more of thefollowing probiotic organisms: Bacteroides uniformis, Collinsellaaerofaciens, Lactobacillus paracasei, Lactobacillus plantarum,Anerostipes caccae, Bacteroides ovatus, Clostridium symbiosum,Streptococcus thermophilus, Lactobacillus reuteri, Pediococcuspentosaceus, Lactobacillus fermentum, Saccharomyces boulardii,Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacteriumbifidum, Lactobacillus helveticus, Bifidobacterium longum, Roseburiahominis, Lactobacillus pentosus, and Lactobacillus rhamnosus. Theseprobiotic organisms may be provided in an approximate CFU range of1.00E+06-1.00E+11.

In one embodiment, the prebiotics in Table 7 may be used in vivo tosupport the following probiotic organisms: Bacteroides uniformis,Bacteroides ovatus, Bacteroides fragilis, Clostridium symbiosum,Collinsella aerofaciens, Roseburia hominis, Anaerostipes caccae,Pediococcus acidilactici, Butyricicoccus pullicaecorum, Roseomonasmucosa, and Dorea longicatena.

TABLE 7 Prebiotic mg/dose Bimuno-galacto-oligosaccharides (B_GOS)500-5000 Mannan-oligosaccharide 500-5000 Resveratrol 100-1000 Hesperetin100-1000 Arabinoxylan-oligosaccharides 500-5000 Dragon fruit 500-5000Beechwood extract xylan 500-5000 Arabinogalactan (Larix laricina)500-5000 DAO antihistamine 500-5000 Xanthan gum 500-5000 Resistantstarch 500-5000 Baobab fruit 100-500  Kongorobi berry 500-5000Resveratrol/Pterostillbene 100-500  Curcumin 100-500  Indian Tinospora(Tinospora cordifolia) (stem and root) 100-500  Poria mushroom 100-500 Nobiletin 500-5000

The amounts in Table 7 may be considered approximations. The prebioticformulation represented in Table 7 may be hereinafter referred to as“Prebiotic Formula Eta.”

In one embodiment, a combination of cultured organisms designed tobenefit the health of the adaptive immune system may include one or moreof the following probiotic organisms: Akkermansia muciniphila,Clostridium symbiosum, Bifidobacterium lactis, Collinsella aerofaciens,Bifidobacterium bifidum, Lactobacillus bulgaricus, Bacteroides fragilis,Bacteroides ovatus, Bifidobacterium animalus, Lactobacillus reuteri, andLactobacillus johnsonii. These probiotic organisms may be provided in anapproximate CFU range of 1.00E+06-1.00E+11.

In one embodiment, a combination of cultured organisms designed tobenefit the health of the innate immune system may include one or moreof the following probiotic organisms: Bacteroides uniformis, Bacilluspumilus, Eubacterium rectale, Faecalibacterium prausnitzii,Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillussalivarus, Streptococcus thermophilus, and Lactobacillus helveticus.These probiotic organisms may be provided in an approximate CFU range of1.00E+06-1.00E+11.

The prebiotics in Table 8 may be used in vivo to support the probioticorganisms listed as benefitting the adaptive and innate immune systems.

TABLE 8 Prebiotic mg/dose Galacto-oligosaccharides 500-5000Mannan-oligosaccharide 500-5000 Resveratrol 100-1000 Hesperetin 100-1000Arabinoxylan-oligosaccharides 500-5000 Dragon fruit 500-5000 Beechwoodextract xylan 500-5000 Arabinogalactan (Larix laricina) 500-5000 DAOantihistamine 500-5000 Xanthan gum 500-5000 Resistant starch 500-5000Baobab fruit 100-500  Kongorobi berry 500-5000Resveratrol/Pterostillbene 100-500  Curcumin 100-500  Indian Tinospora(Tinospora cordifolia) (stem and root) 100-500 

The amounts in Table 8 may be considered approximations. The prebioticformulation represented in Table 8 may be hereinafter referred to as“Prebiotic Formula Theta.”

Certain probiotic organisms may be used to promote or enhancephysiological health more particularly for females, including withoutlimitation, vaginal mucosal maintenance, fertility, and hormonalregulation. In one embodiment, a combination of cultured organismsdesigned to benefit females may include one or more of the followingprobiotic organisms: Lactobacillus salivarius, Bifidobacterium longum,Bifidobacterium breve, Lactobacillus vaginalis, Lactobacillus fermentum,Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus crispatus,Lactobacillus delbrueckii subsp. bulgaricus, Bacteroides uniformis,Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillusplantarum, Lactobacillus jensenii, Bifidobacterium catenulatum, andBifidobacterium pseudocatenulatum. These probiotic organisms may beprovided in an approximate CFU range of 1.00E+06-1.00E+11.

Certain probiotic organisms may be used to promote or enhancephysiological health more particularly for males, including withoutlimitation, urogenital tissue maintenance, fertility, and prostatehealth. In one embodiment, a combination of cultured organisms designedto benefit males may include one or more of the following probioticorganisms: Eubacterium limosum, Lactobacillus paracasei, Bacteroidesthetaiotamicron, Lactobacillus johnsonii, Lactobacillus acidophilus,Bifidobacterium longum, Lactobacillus curvatus, Lactobacillus brevis,Lactobacillus salivarius, Lactobacillus helveticus, Lactobacillus casei,Lactobacillus paracasei, Lactobacillus rhamnosus, and Clostridiumscindens. These probiotic organisms may be provided in an approximateCFU range of 1.00E+06-1.00E+11.

The prebiotics in Table 9 may be used in vivo to support at least thefollowing probiotic organisms: Lactobacillus vaginalis, Eubacteriumlimosum, Bacteroides thetaiotamicron, and Bacteroides uniformis.

TABLE 9 Prebiotic mg/dose Xylo-oligosaccharides 500-5000 Glucomannan(Amorphophallus konjac) 500-5000 Prebiosure WG extract 500-5000Galactomannan 500-5000 Billberry (Vaccinium myrtillus) powder 100-1000Brown seaweed (Alginate fucoidan) 100-1000 Mulberry (Morus nigra)100-1000 Myo-inositol 500-5000 Modified citrus pectin 100-1000 Cassavaroot powder (Manihot esculenta) 100-1000 Glucosamino glycan 500-5000 PHguar gum (Cyamopsis tetragonolobus) 500-5000 Galactomannan Oatoligosaccharide 500-5000 Clitoria ternatea blue pea flower 50-500Cranberry 50-500 Bearberry (Arctostaphylos uva ursi) 50-500

The amounts in Table 9 may be considered approximations. The prebioticformulation represented in Table 9 may be hereinafter referred to as“Prebiotic Formula Iota.”

Generally, the microbiome is formed and maintained by an individual'sdietary intake. Every compound, macronutrient, and micronutrient isprocessed by the microbiome prior to entering the blood stream.Consequently, the microbiome controls caloric response, nutrientresponse, and overall metabolic behavior. Although metabolism iscomplex, a number of organisms are involved in maintaining a healthymetabolism and the absence of these organisms in the gastrointestinaltract leads to markers of unhealthy metabolism and potential weightproblems.

In one embodiment, a combination of cultured organisms designed tobenefit metabolic processes may include one or more of the followingprobiotic organisms: Bacillus clausii, Lactobacillus casei, Akkermansiamuciniphila, Eubacterium rectale, Lactococcus lactis, Lactobacillusbulgaricus, Lactobacillus buchneri, Pediococcus acidilactici,Bifidobacterium longum, Lactobacillus brevis, Lactobacillus delbrueckiisubsp. bulgaricus, Propionibacterium shermanii, Butyricicoccuspullicaecorum, Lactobacillus acidophilus, Lactobacillus rhamnosus GG,Lactobacillus gasseri, Anaerobutyricum hallii, Bifidobacteriumcatenulatum, and Bifidobacterium pseudocatenulatum. These probioticorganisms may be provided in an approximate CFU range of1.00E+06-1.00E+11.

The prebiotics in Table 10 may be used in vivo to support at least thefollowing probiotic organisms: Eubacterium rectale, Akkermansiamuciniphila, Eubacterium hallii, Parabacteroides distasonis,Intestinibacter bartettii, and Christensenella minuta.

TABLE 10 Prebiotic mg/dose Bael fiber 500-5000 Fenugreek fiber 500-5000Organic quinoa sprout 500-5000 Organic chia seed 500-5000 Organic milletsprout 500-5000 Organic buckwheat sprout 500-5000 N-acetyl glucosamineamino glycan 500-5000 Chondroitin 50-500 Mucin 500-5000 Chitosan oligo500-5000 Carrot fiber 500-5000 Promitor (corn fiber)/Nutiose Roquett500-5000 Acemannon 500-5000 Pea fiber 500-5000 Cutch tree (Acaciacatechu)(heartwood and bark)  50-1000 (powder and extract) Pullulan 50-1000 Marshmallow root (Althaea officinalis)  50-1000 Camels milk500-5000

The amounts in Table 10 may be considered approximations. The prebioticformulation represented in Table 10 may be hereinafter referred to as“Prebiotic Formula Kappa.”

Certain probiotic organisms may be used to promote or enhance metabolicprocesses, including without limitation, glucose regulation, ketoneregulation, and energy production. In one embodiment, a combination ofcultured organisms designed to benefit metabolic processes may includeone or more of the following probiotic organisms: Bacillus clausii,Lactobacillus casei, Akkermansia muciniphila, Eubacterium rectale,Faecalibacterium prausnitzii, Bifidobacterium lactis, Lactobacilluslactis, Lactobacillus plantarum, Lactobacillus buchneri, Bacteroidesthetaiotamicron, Bifidobacterium longum, and Lactobacillus brevis. Theseprobiotic organisms may be provided in an approximate CFU range of1.00E+06-1.00E+11.

The prebiotics in Table 11 may be used in vivo to support the probioticorganisms listed as benefitting metabolic processes.

TABLE 11 Prebiotic mg/dose Bael fiber 500-5000 Fenugreek fiber 500-5000Organic quinoa sprout 500-5000 Organic chia seed 500-5000 Organic milletsprout 500-5000 Organic buckwheat sprout 500-5000 N-acetyl glucosamineamino glycan 500-5000 Chondroitin 50-500 Mucin 500-5000 Chitosan oligo500-5000 Carrot fiber 500-5000 Promitor (corn fiber)/Nutiose Roquett500-5000 Acemannon 500-5000 Pea fiber 500-5000 Cutch tree (Acaciacatechu)(heartwood and bark)  50-1000 (powder and extract) Pullulan 50-1000

The amounts in Table 11 may be considered approximations. The prebioticformulation represented in Table 11 may be hereinafter referred to as“Prebiotic Formula Lambda.”

The mouth, esophagus and stomach have a unique microbiome that is notnormally supported by typical probiotics and prebiotics. Certaincultivation and delivery modes are required to support the microbiotafound in the mouth, esophagus and stomach tissues. The design of acombination of probiotics for the mouth, esophagus and stomach utilizesresearch on the upper gastrointestinal microbiota, specific species,growth conditions and requirements followed by differentialstabilization in a powder dosage form.

In one embodiment, a combination of cultured organisms designed tobenefit the mouth, esophagus and stomach may include one or more of thefollowing probiotic organisms: Streptococcus uberis, Streptococcusoralis, Streptococcus rattus, Streptococcus salivarius, Saccharomycescerevisiae, Bacillus coagulans, Mycobacterium vaccae, Lactobacillussalivarius, Lactobacillus helveticus, Lactobacillus fermentum,Bifidobacterium lactis, Enterococcus faecalis, Lactobacillus kefiri, andButyricicoccus pullicaecorum. These probiotic organisms may be providedin an approximate CFU range of 1.00E+06-1.00E+11.

In one embodiment, a combination of cultured organisms designed tobenefit the mouth, esophagus and stomach may include one or more of thefollowing probiotic organisms: Bifidobacterium lactis, Lactobacillusbrevis, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillussalivarius, Streptococcus salivarius, Lactobacillus casei, Lactobacillusacidophilus, Lactobacillus plantarum, Streptococcus uberis (KJ2®),Streptococcus oralis (KJ3®), and Streptococcus rattus (JH145®). Theseprobiotic organisms may be provided in an approximate CFU range of1.00E+06-1.00E+11.

In one embodiment, a combination of cultured organisms designed tobenefit the endocrine system, autocrine, may include one or more of thefollowing probiotic organisms: Bacteroides uniformis, Eubacteriumlimosum, Bacillus indicus, Lactobacillus iners, Bacillus megaterium,Lactobacillus johnsonii, Lactobacillus delbrueckii subsp. bulgaricus,Lactobacillus acidophilus, Bacillus coagulans, and Bacteroidesthetaiotamicron. These probiotic organisms may be provided in anapproximate CFU range of 1.00E+06-1.00E+11.

In one embodiment, a combination of cultured organisms designed tobenefit the endocrine system, paracrine, may include one or more of thefollowing probiotic organisms: Bacillus coagulans, Bifidobacteriumlongum, Bifidobacterium breve, Eubacterium limosum, Lactobacillusfermentum, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillusvaginalis, Lactobacillus rhamnosus A, Lactobacillus crispatus,Lactobacillus iners, and Lactobacillus delbrueckii subsp. bulgaricus.These probiotic organisms may be provided in an approximate CFU range of1.00E+06-1.00E+11.

The prebiotics in Table 12 may be used in vivo to support the probioticorganisms listed as benefitting the endocrine system.

TABLE 12 Prebiotic mg/dose Glycomacropeptide 500-5000Xylo-oligosaccharides 500-5000 Glucomannan (Amorphophallus konjac)500-5000 Prebiosure WG extract 500-5000 Galactomannan 500-5000 Blueberry100-1000 Thymus 100-1000 Mulberry (Morus nigra) 100-1000 Myo-inositol500-5000 Modified citrus pectin 100-1000 Tapioca 100-1000 Glucosaminoglycan 500-5000 PH guar gum (Cyamopsis tetragonolobus) 500-5000Galactomannan Oat oligosaccharide 500-5000 Clitoria ternatea blue peaflower 50-500 Cranberry 50-500 Bearberry (Arctostaphylos uva ursi)50-500

The amounts in Table 12 may be considered approximations. The prebioticformulation represented in Table 12 may be hereinafter referred to as“Prebiotic Formula Mu.”

Certain probiotics may be more beneficial for children because there areso many developmental processes that rely on a healthy microbiome, andthey need a constant supply of healthy prebiotic oligosaccharides.

In one embodiment, a combination of cultured organisms designed tobenefit children may include one or more of the following probioticorganisms: Bifidobacterium animalus, Bifidobacterium infantis,Bifidobacterium adolescentis, Lactobacillus gasseri, Lactococcus lactis,Bifidobacterium longum pseudo, Lactobacillus bulgaricus, and Bacteroidesovatus. These probiotic organisms may be provided in an approximate CFUrange of 1.00E+06-1.00E+11.

The prebiotics in Table 13 may be used in vivo to support the probioticorganisms listed as benefitting children.

TABLE 13 Prebiotic mg/dose 2′frucosyllactose 500-5000Isomalto-oligosaccharide 500-5000 Fructo-oligosaccharide 500-5000Galacto-oligosaccharide 500-5000 Rutin 100-1000 Oat oligolactolipids500-5000 Sialylated bovine milk oligosaccharides (S-BMO) 100-5000IgY-immunoglobulin (IgY MAX ®) 25-500 Beta glucan 100-1000 Maca 100-1000Lacto-oligosaccharide 100-1000 Medium chain triglycerides 500-5000Phosphatidyl choline 500-5000 Rose hips 500-5000

The amounts in Table 13 may be considered approximations. The prebioticformulation represented in Table 13 may be hereinafter referred to as“Prebiotic Formula Nu.”

Fermented probiotics can provide substantial health benefits, butferments often contain contaminants and can affect the gastrointestinaltract in negative ways. Providing a substantially pure combination ofprobiotics in a stable, powder form can provide all the benefits offermented organisms and their metabolites, with the complications thatcome from contaminants. For example and not by way of limitation, astable, powder product may include freeze-dried kefir, freeze-driedkombucha sobas, DAO (diamineoxidase) porcine, and kidney proteinconcentrate.

In one embodiment, a combination of cultured organisms that may beincluded in a stable, powder product and designed to provide thebenefits of fermented organisms and their metabolites may include one ormore of the following probiotic organisms: Bacillus pumilus,Desulfovibrio piger, Lactococcus lactis, Lactobacillus sakei,Propionibacterium freundenreichii, Lactobacillus helveticus, andSaccharomyces cerevisiae. These probiotic organisms may be provided inan approximate CFU range of 1.00E+06-1.00E+11.

The subject invention may be more easily comprehended by reference tothe specific embodiments recited herein, which are representative of theinvention. However, it must be understood that the specific embodimentsare provided only for the purpose of illustration, and that theinvention may be practiced in a manner separate from what isspecifically illustrated without departing from its scope and spirit.

What is claimed and desired to be secured by United States LettersPatent is:
 1. A method for a probiotic product designed to promote andmaintain immune system function comprising: selecting at least oneprobiotic organism from the group consisting of Collinsella aerofaciens,Anaerostipes caccae, Bacteroides ovatus, Clostridium symbiosum, andRoseburia nominis; selecting a growth broth for growing the probioticorganism, wherein the growth broth includes a prebiotic formula that isoptimized for growing the probiotic organism; inoculating the growthbroth with the probiotic organism; growing the probiotic organism in thegrowth broth; harvesting the probiotic organism from the growth broth;selecting an agar spread plate for growing the probiotic organism,wherein the agar spread plate media includes a prebiotic formula that isoptimized for growing the probiotic organism; inoculating the agarspread plate with the probiotic organism harvested from the growthbroth; growing the probiotic organism on the agar spread plate;harvesting the probiotic organism from the agar spread plate;freeze-drying the probiotic organism; and including the probioticorganism and an effective amount of Prebiotic Formula Eta in theproduct.
 2. The method of claim 1, further comprising: freeze-drying theprobiotic organism harvested from the agar spread plate by use of alyophilization reagent, wherein the lyophilization reagent comprisesmannitol, trehalose, glycerol, cysteine, ascorbic acid, glutathione,uric acid, and activated charcoal.
 3. The method of claim 1, wherein theagar spread plate media is comprised of approximately 40 g/L tryptic soyagar, approximately 50 ml/L defibrinated sheep blood, approximately 0.5g/L cysteine, and approximately 0.1 g/L glutathione.
 4. The method ofclaim 1, wherein the at least one probiotic organism to grow is selectedfrom the group consisting of Bacteroides ovatus and Anaerostipes caccae,and the prebiotic formula included in the growth broth and in the agarspread plate media is Prebiotic Formula Gamma.
 5. The method of claim 1,wherein two probiotic organisms, Bacteroides ovatus and Anaerostipescaccae, are selected to grow and the prebiotic formula included in thegrowth broth and in the agar spread plate media is Prebiotic FormulaGamma.
 6. The method of claim 1, wherein the at least one probioticorganism to grow is selected from the group consisting of Collinsellaaerofaciens, Bacteroides ovatus, Clostridium symbiosum, and Roseburiahominis, and the prebiotic formula included in the growth broth and inthe agar spread plate media is Prebiotic Formula Delta.
 7. A method fora probiotic product designed to promote and maintainmicrobiota-gut-brain axis health comprising: selecting at least oneprobiotic organism from the group consisting of Enterococcus faecium,Bifidobacterium adolescentis, Faecalibacterium prausnitzii,Mycobacterium vaccae, Lactobacillus plantarum, Bifidobacterium longum,Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillusfarciminis, Propionibacterium shermanii, Bifidobacterium infantis,Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillusdelbrueckii, Bacteroides fragilis, Bifidobacterium lactis, andLactobacillus rhamnosus A; selecting a growth broth for growing theprobiotic organism, wherein the growth broth includes a prebioticformula that is optimized for growing the probiotic organism;inoculating the growth broth with the probiotic organism; growing theprobiotic organism in the growth broth; harvesting the probioticorganism from the growth broth; selecting an agar spread plate forgrowing the probiotic organism, wherein the agar spread plate mediaincludes a prebiotic formula that is optimized for growing the probioticorganism; inoculating the agar spread plate with the probiotic organismharvested from the growth broth; growing the probiotic organism on theagar spread plate; harvesting the probiotic organism from the agarspread plate; freeze-drying the probiotic organism; and including theprobiotic organism and an effective amount of Prebiotic Formula Zeta inthe product.
 8. The method of claim 7, further comprising: freeze-dryingthe probiotic organism harvested from the agar spread plate by use of alyophilization reagent, wherein the lyophilization reagent comprisesmannitol, trehalose, glycerol, cysteine, ascorbic acid, glutathione,uric acid, and activated charcoal.
 9. The method of claim 7, wherein thefreeze-drying reagent is Freeze-drying Buffer E when the probioticorganism is Faecalibacterium prausnitzii.
 10. The method of claim 7,wherein the probiotic organism is selected from the group consisting ofFaecalibacterium prausnitzii, and Mycobacterium vacae, and the prebioticformula included in the growth broth and in the agar spread plate mediais Prebiotic Formula Alpha.
 11. The method of claim 7, wherein theprobiotic organisms grown include Faecalibacterium prausnitzii andMycobacterium vacae and the prebiotic formula included in the growthbroth and in the agar spread plate media is Prebiotic Formula Alpha. 12.The method of claim 7, wherein the probiotic organism is Bacteroidesfragilis and the prebiotic formula included in the growth broth and inthe agar spread plate media is Prebiotic Formula Delta.
 13. The methodof claim 10, wherein the product promotes a reduction of anxiety andstress.
 14. A method for a probiotic product designed to promote andmaintain health for a female comprising: selecting at least oneprobiotic organism from the group consisting of Lactobacillussalivarius, Bifidobacterium longum, Bifidobacterium breve, Lactobacillusvaginalis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillusreuteri, Lactobacillus crispatus, Lactobacillus delbrueckii subsp.bulgaricus, Bacteroides uniformis, Lactobacillus rhamnosus,Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillusjensenii, Bifidobacterium catenulatum, and Bifidobacteriumpseudocatenulatum; selecting a growth broth for growing the probioticorganism, wherein the growth broth includes a prebiotic formula that isoptimized for growing the probiotic organism; inoculating the growthbroth with the probiotic organism; growing the probiotic organism in thegrowth broth; harvesting the probiotic organism from the growth broth;selecting an agar spread plate for growing the probiotic organism,wherein the agar spread plate media includes a prebiotic formula that isoptimized for growing the probiotic organism; inoculating the agarspread plate with the probiotic organism harvested from the growthbroth; growing the probiotic organism on the agar spread plate;harvesting the probiotic organism from the agar spread plate;freeze-drying the probiotic organism; and including the probioticorganism and an effective amount of Prebiotic Formula Iota in theproduct.
 15. The method of claim 14, wherein the at least one probioticorganism to grow is selected from the group consisting of Lactobacillusvaginalis, and Bacteroides uniformis, and the prebiotic formula includedin the growth broth and in the agar spread plate media is PrebioticFormula Beta.
 16. The method of claim 15, wherein the product promoteshormonal regulation.
 17. The method of claim 15, wherein the productpromotes fertility.